An object of the present invention is to mass-produce canine interleukin 12 (hereinafter sometimes abbreviated xe2x80x9cCaIL12xe2x80x9d) with the primary structure of its protein derived from canine genetic information for providing a drug for treatment of animals. Such drugs may be antitumor and antiviral in nature and can act as a vaccine adjuvant. The present invention relates to DNA sequences, recombinant vectors and transformants, CaIL12 itself and a production process thereof.
The present invention also relates to an immune disease remedy and preventive agent for dogs and cats comprising canine interleukin 12 with the primary structure of its protein derived from canine genetic information, and an immune disease treatment method and preventive method for dogs and cats using the remedy or preventive agent.
Interleukin 12 is a heterodimer consisting of a protein with a molecular weight of about 40 kD (hereinafter sometimes abbreviated xe2x80x9cP40xe2x80x9d or xe2x80x9cP40 subunitxe2x80x9d) and a protein with a molecular weight of about 35 kD (hereinafter sometimes abbreviated xe2x80x9cP35xe2x80x9d or xe2x80x9cP35 subunitxe2x80x9d), and is a cytokine with such bioactivity as to activate natural killer cells and type 1 helper T cells, being abbreviated as IL12. Concerning IL12, some literature has been published. In addition to human IL12, the cDNAs of mouse IL12 (Shoenhaut et al: J. Immunol. 148, 3433-3440 (1992) and bovine IL12 (GenBank Database Registration Nos. U11815 and U14416) were cloned by gene manipulation techniques, and their application as remedies against cancers, viral diseases, etc. are being researched and developed.
For IL12, treatment effects in mouse model experiments against tumors, infectious diseases, allergies, and the like especially by its activity in boosting the cellxe2x80x94mediated immune response have been reported. in the literature such as Nastala et al: J. Immunol. 153, 1697-1706 (1994), Gazzinelli et al: Proc. Natl. Acad. Sci. USA 90, 6115-6119 (1993) and Chirgwin et al: Biochemistry 18, 5294 (1979), and clinical trials of IL12 as a remedy against human cancer and human infectious disease has already started. However, it is not yet reported that canine IL12 has been cloned.
Dogs are known to suffer from various cancers such as mammary gland tumor, various viral diseases such as Parvovirus infectious disease and distemper infectious disease, allergic dermatitis and the like. Therefore, if canine IL12 were to become easily available, it is expected that its application as a canine antitumor drug, antiallergic drug and antiviral drug would be attempted.
Presently, few remedies and preventive agents effective against these canine diseases are available. For example, most dogs suffering from cancer come to hospitals after their tumors have grown, and even if the tumors are ablated by surgery, they soon die after the operation because of metastasis. Also, for skin diseases often seen with dogs, they cannot be cured in most cases even if steroids are administered repetitively for long periods of time as a treatment. As a consequence, fast and continuously acting remedies are being demanded. It is expected that new applications will be attempted for these canine diseases now left without any effective remedy if canine IL12 becomes available.
The invention includes cloning CaIL12 cDNA for mass-production and for providing a preparation containing CaIL12 as a remedy or preventive agent for dogs and cats suffering from immune diseases. The invention particularly includes cloning cDNAs coding for P40 and P35 of CaIL12 from canine cDNA and, furthermore, producing cells capable of producing CaIL12 using two expression plasmids linked to these cDNAs, respectively, and in producing recombinant Baculovirus containing both the genes.
Thus, the invention has established a method for mass-producing CaIL12 and found that if a preparation containing CaIL12 is administered to dogs and cats suffering from diseases difficult to treat by conventional therapeutic methods or if lymphocytes isolated from the peripheral blood of a sick dog or cat are stimulated in vitro by a preparation containing CaIL12 and returned into the body of the dog or cat again, the disease can be surprisingly remarkably improved.
The present invention provides plasmids capable of producing CaIL12, a transformant of Escherichia coli having these plasmids, animal cells transformed by these plasmids, CaIL12 obtained from these transformants, Baculovirus capable of producing CaIL12, CaIL12 produced by the Baculovirus, an immune disease remedy and preventive agent for dogs and cats containing CaIL12 as a main effective ingredient, and an immune disease treatment method and preventive method for dogs and cats. In addition, the present invention also provides cDNAs respectively coding for two subunits of CaIL12 protein.
Important aspects of the present invention include:
(1) Canine interleukin 12 comprises a P40 subunit and a P35 subunit and has at least one ability selected from 1) an ability to induce an antiviral active factor acting on canine leukocytes and a factor to intensify the expression of class II mouse histocompatability antigens (MHC) of canine tumor cells, 2) an ability to promote the proliferation of canine lymphoblasts, and 3) an ability to damage canine tumor cells by activating canine leukocytes.
(2) Canine interleukin 12, stated in paragraph (1) above, wherein the P40 subunit has an amino acid sequence substantially the same as SEQ ID NO:1 or SEQ ID NO:11 or corresponding to part of either of those sequences.
(3) Canine interleukin 12, stated in paragraph (1) above, wherein the P35 subunit has an amino acid sequence substantially the same as SEQ ID NO:2 or SEQ ID NO:12 or corresponding to part of either of those sequences.
(4) An immune disease remedy and preventive agent for dogs and cats comprising canine interleukin 12 and a treatment method and preventive method using the remedy or preventive agent.
(5) A DNA sequence coding for the canine interleukin 12 stated in any one of paragraphs (1), (2) and (3) above, a recombinant vector containing it, a transformant obtained by transforming host cells by the recombinant vector, and a method of producing canine interleukin 12 by culturing the transformant or infecting insect cells or larvae with the transformant for culturing or raising.
SEQ ID No:1 sets forth the the P40 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID No:2 sets forth the nucleotide sequence encoding the P35 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID NO:3 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P40 cDNA.
SEQ ID NO:4 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P40 cDNA.
SEQ ID NO:5 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P35 cDNA.
SEQ ID NO:6 sets forth the nucleotide sequence for one of the primers used in the cloning of CaIL12 P35 cDNA.
SEQ ID NO:7 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:8 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:9 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:10 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID No:1 sets forth the nucleotide sequence encoding the P40 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID No:2 sets forth the nucleotide sequence encoding the P35 subunit of canine interleukin 12 and the corresponding amino acid sequence.
SEQ ID NO:13 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:14 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:15 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
SEQ ID NO:16 sets forth the nucleotide sequence for a primer used in the preparation of a CaIL12 expression vector.
Plasmids in which DNAs coding for the two subunits of CaIL12 protein of the present invention can be produced, for example, are described below. Two genes respectively coding for the two subunits showing CaIL12 activity can be cloned by polymerase chain reaction (hereafter sometimes abbreviated xe2x80x9cPCRxe2x80x9d) of cDNAs synthesized after extracting poly(A)RNA from canine cells, using primers based on the gene sequences respectively coding for the two subunits of bovine or human IL12. As another method, the total length of CaIL12 P40 cDNA and CaIL12 P35 cDNA can be cloned by plaque hybridization of a phage library prepared from a synthesized cDNA recombinant, with two cDNA fragments obtained by PCR.
Usual methods can be used to obtain RNA from a canine organ or cells. For example, canine monocytes or lymphocytes stimulated by a mitogen or the like may be used. Such methods include isolating polysomes or using sucrose density gradient centrifugation or electrophoresis. RNA can be extracted from the canine organ or cells by any proper method selected from the guanidine thiocyanate cesium chloride method to effect CsCl density gradient centrifugation after guanidine thiocyanate treatment as disclosed by Nastala et al: J. Immunol. 153, 1697-1706 (1994), phenol extraction after treatment by a surfactant in the presence of ribonuclease inhibitor using a vanadium composite as disclosed by Gazzinelli et al: Proc. Natl. Acad. Sci. USA 90, 6115-6119 (1993), guanidine thiocyanate hot phenol method, guanidine thiocyanate guanidine hydrochloric acid method, guanidine thiocyanate phenol chloroform method, precipitation of RNA by treatment with lithium chloride after treatment with guanine thiocyanate and the like.
mRNA is isolated from a canine organ or canine monocytes or lymphocytes stimulated with a mitogen or the like by any ordinary method such as lithium chloride urea method, guanidine isocyanate method, or oligo dT cellulose column method, etc. A cDNA is synthesized from the obtained mRNA by any ordinary method such as Gubler et al.""s method or H. Okayama et al.""s method (Chirgwin et al: Biochemistry 18, 5294 (1979)). A reverse transcriptase such as avian myeloblastosis virus (AMV) is used to synthesize a cDNA from the obtained mRNA, as required in combination with DNA polymerase, etc. using a primer. However, it is convenient to use a marketed synthesizing or cloning kit.
cDNAs coding for the P40 subunit and P35 subunit showing CaIL12 activity can be cloned if PCR is effected with cDNAs as templates using a primer based on a human, mouse or bovine base sequence. As another method, synthesized cDNAs are linked with xcex phage vectors, and mixed in vitro with a xcex phage coated protein, etc., for packaging. Escherichia coli, acting as a host, are infected with the produced phage particles. In this case, the Escherichia coli infected with a xcex phage are lysed and individual clones are collected as plaques. The plaques are transferred onto a filter of nitrocellulose or the like and by hybridization using the radio labeled genes obtained by PCR as a probe, the full length of CaIL12 P40 cDNA and CaIL12 P35 cDNA can be cloned.
A procaryote or eucaryote can be used as a host. The procaryotes which can be used here include bacteria, especially Escherichia coli or Bacillus such as Bacillus subtilis. The eucaryotes which can be used here include eucaryotic microbes such as yeasts for example, Saccharomyces such as Saccharomyces cerevisiae, insect cells such as Spodoptera frugiperda cells, Trichoplusiani cells and Bombyx mori cells, and animal cells such as human cells, simian cells and mouse cells. In the present invention, organisms themselves, for examples, insects such as Trichoplusiani can also be used.
The expression vectors which can be used here include plasmids, phages, phagemids, viruses (Baculovirus), insects, vaccinia (animal cells) and the like. The promoter in the expression vector is selected depending on the host cells. For example, promoters for bacteria include lac promoter, trp promoter and the like, and promoters for yeasts include adhl promoter, pqk promoter and the like. Promoters for insects include Baculovirus polyhedrin promoter, p10 promoter and the like. Promoters for animal cells include early or late promoter of Simian Virus 40 and the like. However, the promoters which can be used are not limited to the above.
The transformation of a host by an expression vector can be effected by any conventional method well known to persons skilled in the art. These methods are stated, for example, in Current Protocols in Molecular Biology, John Wiley and Sons. Culturing transformants can also be effected according to conventional methods.
The produced CaIL12 has an apparent molecular weight of about 70 to 80 kD, if determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The 70-80 kD band in SDS-PAGE produces two subunits of about 40 kD and about 35 kD in molecular weight under reducing conditions.
CaIL12 is mainly characterized by its ability to induce canine interferon xcex3 from canine leukocytes and the effect of promoting the proliferation of canine lymphocytes stimulated with various mitogens such as phytohemagglutinin (hereinafter sometimes abbreviated as xe2x80x9cPHAxe2x80x9d). It also has activity to activate NK cells and cytotoxic T cells for killing their target cells, for example, the cell line derived-from a tumor or fibroblasts infected with a virus.
The immune disease remedy and preventive agent for dogs and cats of the present invention shows a surprising remarkable treatment effect and preventive effect against various immune diseases of dogs and cats such as diseases declining in immunological competence such as tumors, dermatitis, allergic diseases and infectious diseases, and diseases showing partial immune reaction mainly relying on humoral reaction rather than on cellular immune reaction, compared to conventional remedies and preventive agents and treatment methods and preventive methods against these canine diseases.
The tumors of dogs and cats include mammary gland tumor, eosinophilic granuloma, epidermoid, ecphyma, lipoma, othematoma, pulmonary edema, dermal caulescent soft tumor, anal tumor and the like. Dermatitis of dogs and cats includes external acoustic meatus inflammation, dermatitis, eczema, dermatomycosis, pyoderma, allergic dermatitis, urtication, traumatic dermatitis and alopecia. Infectious diseases of dogs and cats include canine Parvovirus, distemper, feline AIDS, feline leukemia and the like. Allergic diseases include canine and feline pollinosis.
The immune remedy and preventive agent for dogs and cats can also contain other arbitrary ingredients in addition to CaIL12. The ingredients added to these medicines are mainly decided in reference to the medicine administration method. When the medicine is used as a solid, a filler such as lactose, binder such as carboxymethyl cellulose or gelatin, colorant and coating agent and the like can be used. Such a formulation is suitable for oral administration.
White Vaseline(copyright), cellulose derivatives, surfactants, polyethylene glycol, silicone, olive oil and the like can be added as a solid or activator, to prepare a cream, emulsion, lotion and the like for application to the diseased part as an external medicine.
When the medicine is administered as a liquid, it can contain a physiologically permissible solvent, emulsifier and stabilizer as usually practiced. The solvent can be water or isotonic physiological salt solution such as PBS, and the emulsifier can be a polyoxyethylene based surfactant, fatty acid based surfactant, silicone or the like. The stabilizer can be canine serum albumin, a polyol such as gelatin or a saccharide such as sorbitol or trehalose. The method for administering the remedy and preventive agent of the present invention is not especially limited, but injection, is expected to give the highest treatment effect. The forms of injection method are not limited. They include intravenous injection, intramuscular injection, hypodermic injection, intraperitoneal injection or intrapleural injection. The dosage is decided in reference to the size of the solid, administration method, disease concerned, symptom, etc., and an amount sufficient to manifest the treatment and/or preventive effect can be administered. For example, administration of CaIL12 by 0.1 pg to 100 xcexcg/kg per day can provide a sufficient therapeutic effect.
In the case of adoptive immunotherapy, if 1 to 100 ml of lymphocytes isolated from canine blood are stimulated by 0.001 pg to 1 xcexcg/kg of CaIL12 for 12 hours to 6 days and returned into the body again, a sufficient effect can be obtained.